CN104845995A - Method for dynamic regulation and control of threonine efflux transport protein gene expression for production of L-threonine - Google Patents
Method for dynamic regulation and control of threonine efflux transport protein gene expression for production of L-threonine Download PDFInfo
- Publication number
- CN104845995A CN104845995A CN201410050917.2A CN201410050917A CN104845995A CN 104845995 A CN104845995 A CN 104845995A CN 201410050917 A CN201410050917 A CN 201410050917A CN 104845995 A CN104845995 A CN 104845995A
- Authority
- CN
- China
- Prior art keywords
- threonine
- sequence
- expression element
- gene
- gene expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for dynamic regulation and control of threonine efflux transport protein gene expression for production of L-threonine, the present invention provides a method to construct recombinant bacteria, the method includes the following steps: the recombinant bacteria is obtained by introduction of mutant threonine operon thrAC1034TBC expression element, repressor protein gene expression element, and threonine efflux gene expression element into target bacteria; and the threonine efflux gene expression element includes threonine efflux gene, an induced promoter to drive the gene expression, and control component Ocmt between the threonine efflux gene and the induced promoter. The beneficial effects are that according to the specific effects of gene expression on threonine production of fermentation logarithmic phase and stable phase, a threonine efflux transporter protein gene expression control strategy is specifically put forward, and a new threonine fermentation amount improvement way is disclosed and proved by practice, so that in practice the method can be used for bacterial fermentation for production of threonine, and is convenient to popularize and apply.
Description
Technical field
The present invention relates to biological technical field, belong to field of amino acid fermentation, be specifically related to a kind of method that dynamic regulation Threonine arranges transporter gene Expression product L-threonine outward.
Background technology
L-threonine is a kind of essential amino acid, is widely used in the aspects such as medicine, food, animal-feed.In recent years, the demand of domestic and international market to L-threonine increases year by year.
Fermentation method is the main method of current suitability for industrialized production L-threonine, and the structure of high productivity energy bacterial classification is the key of fermentative production Threonine.In the process that bacterial classification builds, the method for process LAN threonine operon is generally adopted to strengthen the route of synthesis of Threonine.When in born of the same parents, production amount of threonine runs up to finite concentration, in born of the same parents, the Threonine meeting feedback repression of high density and the key enzyme of suppression threonine synthetic pathway, even activate the degradation pathway of Threonine.The outer row of Threonine then becomes the step that restriction Threonine efficiently synthesizes, and therefore needs to accelerate Threonine and transports outside born of the same parents, improve the Threonine production performance of bacterial classification further.In disclosed document, identify multiple Threonine arrange transporter gene outward, as colibacillary Threonine efflux protein gene rhtA, rhtB and rhtC, the thrE gene etc. of Corynebacterium glutamicum.In published Threonine Fermentation production technology, arrange by the above-mentioned Threonine of simple process LAN the output that transporter gene can improve Threonine outward.
Summary of the invention
An object of the present invention is to provide a kind of method building recombinant bacterium.
Method provided by the invention is following A or B:
Method shown in A comprises the steps: that repressor protein gene Expression element and Threonine being arranged gene expression element importing outward sets out in bacterium, obtains recombinant bacterium;
Method shown in B comprises the steps: repressor protein gene Expression element, Threonine to arrange gene expression element and MUTATION Threonine operon Expression element outward and imports and set out in bacterium, obtains recombinant bacterium;
Described Threonine is arranged gene expression element outward and is comprised the control region that Threonine arranges gene outward, drives the Threonine inducible promoter of arranging genetic expression outward and be positioned at therebetween;
The promotor that described repressor protein gene Expression element comprises repressor protein gene and drives repressor protein gene to express;
The promotor that described MUTATION Threonine operon Expression element comprises MUTATION Threonine operon and drives MUTATION Threonine operon to express.
In aforesaid method,
In method shown in B, described lead-in mode is following 1) or 2):
1) described repressor protein gene Expression element, described Threonine are arranged gene expression element and described MUTATION Threonine operon Expression element outward to be imported by recombinant vectors A and set out in bacterium, obtain recombinant bacterium;
2) described repressor protein gene Expression element and described Threonine being arranged gene expression element outward sets out in bacterium by recombinant vectors B importing, and set out in bacterium described in described MUTATION Threonine operon Expression element is imported by homologous recombination, obtain recombinant bacterium.
In aforesaid method, 1) in, described repressor protein gene Expression element, described Threonine are arranged the carrier obtained in gene expression element and described MUTATION Threonine operon Expression element insertion expression vector outward by described recombinant vectors A;
2), in, described repressor protein gene Expression element and described Threonine are arranged gene expression element outward to insert the carrier obtained in expression vector by described recombinant vectors B;
Described homologous recombination is set out in bacterium to described with the form homologous recombination of the fragment containing MUTATION Threonine operon Expression element by MUTATION Threonine operon Expression element.
In aforesaid method, it is rhtC or rhtC and rhtB or rhtC, rhtB and rhtA that described Threonine arranges gene outward;
The inducible promoter that described driving Threonine arranges genetic expression is outward P
t5;
Described control region is Ocmt;
The promotor that described driving repressor protein gene is expressed is P
km;
Described MUTATION Threonine operon is MUTATION Threonine operon thrA
c1034TbC
The promotor that described driving MUTATION Threonine operon is expressed is P
pL.
In aforesaid method, described promotor P
t5nucleotides sequence be classified as sequence 3 or 4 or 5 in sequence table from 5 ' end 1-59 position Nucleotide;
The nucleotides sequence that described Threonine arranges gene rhtC is outward classified as the sequence 5 of sequence 3 in the sequence table sequence 4 in 5 ' end 92-835 position Nucleotide or sequence table in 5 ' end 92-733 position Nucleotide or sequence table from 5 ' end 92-733 position Nucleotide;
The nucleotides sequence that described Threonine arranges gene rhtA is outward classified as sequence 4 in the sequence table sequence 5 in 5 ' end 734-1706 position Nucleotide or sequence table from 5 ' end 734-1637 position Nucleotide;
The nucleotides sequence that described Threonine arranges gene rhtB is outward classified as sequence 5 in sequence table from 5 ' end 1638-2342 position Nucleotide;
The nucleotides sequence of described Ocmt is classified as sequence 3 or 4 or 5 in sequence table from 5 ' end 60-91 position Nucleotide;
Described promotor P
kmnucleotides sequence be classified as sequence 2 in sequence table from 5 ' end 1-144 position Nucleotide;
The nucleotides sequence of described repressor protein gene is classified as sequence 2 in sequence table from 5 ' end 145-917 position Nucleotide;
Described MUTATION Threonine operon thrA
c1034Tthe nucleotides sequence of BC is classified as sequence 1 in sequence table from 5 ' end 688-5654 position Nucleotide;
Described promotor P
pLnucleotides sequence be classified as sequence 1 in sequence table from 5 ' end 520-687 position Nucleotide.
In aforesaid method, the nucleotides sequence of described repressor protein gene Expression element is classified as the sequence 2 in sequence table;
The nucleotides sequence that described Threonine arranges gene expression element is outward classified as sequence 3 in sequence table or sequence 4 or sequence 5;
The nucleotides sequence of described MUTATION Threonine operon Expression element is classified as sequence 1 in sequence table from 5 ' end 520-5654 position Nucleotide;
The nucleotides sequence of the described fragment containing MUTATION Threonine operon Expression element is classified as the sequence 1 in sequence table.
In aforesaid method, described in set out bacterium for knocking out the metA gene of encoded homoserine succinyltransferase in bacterium (homoserine O-succinyltransferase), the ilvA gene of encoding Thr desaminase (threoninedeaminase), the lysA gene of encoding D AP decarboxylase (diaminopimelate decarboxylase), the tdh gene of encoding Thr dehydratase (threonine dehydrogenase), encoding Thr absorption and transport albumen (also can be called serine/threonine:H
+symporter) tdcC gene and encoding Thr absorption and transport albumen (also can be called serine/threonine:Na
+symporter) sstT gene is the bacterium that obtains of totally 6 kinds of genes.
In an embodiment of the present invention, the bacterium that sets out described in is knock out metA gene, ilvA gene, lysA gene, tdh gene, tdcC gene and the sstT gene bacterium that obtains of totally 6 kinds of genes successively.
Above-mentioned bacterium is Escherichia bacteria, is more preferably intestinal bacteria, as the follow-up bacterial strain of e. coli k-12 bacterial strain, comprises the bacterial strain that W3110 is derivative.The recombinant bacterium prepared by aforesaid method is also the scope of protection of the invention.
Another object of the present invention is to provide a kind of method generating Threonine.
Method provided by the invention, comprises the steps: the recombinant bacterium that fermentation culture is above-mentioned, and carries out cuminic acid induction at the second growth phase of described recombinant bacterium, collects the supernatant liquor of tunning, obtains Threonine;
Described second growth phase is exponential growth later stage, stationary phase or decline phase.
The described exponential growth later stage is 13-15 hour from fermentation culture;
Described stationary phase is 16-50 hour from fermentation culture;
Described decline phase is 50-96 hour from fermentation culture.
In aforesaid method, described cuminic acid induction is 5-1000 μm of ol/L cuminic acid for adding final concentration in fermentation system;
The induction of described cuminic acid is specially that in fermentation system, add final concentration be 5-500 μm of ol/L cuminic acid.
Described method also comprises the steps: to add glucose in described fermentation culture, makes the concentration of glucose in fermentation system maintain 10 ± 5g/L; And be that 0.6:1.0:1.3 adds methionine(Met), Isoleucine and Methionin according to mass ratio in described fermentation culture, make thalline specific growth rate maintain 0.12-0.2h
-1.
In an embodiment of the present invention, carry out cuminic acid induction in the stationary phase of described recombinant bacterium, in the 16th hours fermentation system from fermentation culture, namely add final concentration is that 100 μm of ol/L cuminic acid are induced.
Or a kind of recombinant vectors, be the recombinant vectors in aforesaid method.
In the present invention, fermentation mode can make batch fermentation, fed-batch fermentation, semicontinuous fermentation or continuously ferment (microbial project technological principle, Yao Ru Huazhoushishui), is preferably fed-batch fermentation.In the ideal situation, the exemplary fermentation curve of microorganism in liquid fermentation medium comprises lag phase, exponential phase, stationary phase and decline phase (microbiology study course, Zhou Deqing; Microbiology, I.E. Acker is not shown).In the fermenting process of reality, between exponential phase and stationary phase, there is the stage that thalline slowly grows, be called the exponential growth later stage.In the present invention, term " subordinate phase of fermenting process " comprises exponential growth later stage, stationary phase and decline phase, and term " first stage of fermenting process " refers to the time period before " subordinate phase of fermenting process ", comprise lag phase and exponential phase of growth.In the present invention, " Threonine of the bacterium that transformation is obtained is arranged transporter gene outward and do not expressed or low amounts expression in the first stage of fermenting process " does not add or add low amounts inductor by any one or more time points of the first stage at fermenting process to realize; " the second-order great expression at fermenting process " adds the inductor realization of high density by any one or more time points of the subordinate phase at fermenting process.
Experiment of the present invention proves, the present invention adopts cuminic acid (cuminic acid) abduction delivering element regulation and control Threonine to arrange the expression of gene outward, the ultimate principle of this abduction delivering element regulator control system is: the aporepressor of the cymR genes encoding of Pseudomonas putidas F1 and the regulatory gene Ocmt of cmt operon interact, and stops the Transcription of RNA polymerase.When there is cuminic acid in born of the same parents, after aporepressor is combined with cuminic acid, just lose the ability with the combination of Ocmt sequence, rna polymerase transcribe is proceeded.Cuminic acid regulates and controls the expression of this transcription unit as inductor.Use colibacillary weak promoter P
kmcontrol cymR genetic expression, prevents the aporepressor of synthesis high density in born of the same parents from producing toxicity (Choi, Y.J., et al.Applied and Environmental Microbiology, 2010.76 (15): 5058-5066.) to Host Strains.Use colibacillary strong promoter PT5 to be connected with regulating and controlling sequence Ocmt, after regulating and controlling sequence, connect Threonine efflux protein gene rhtA further, rhtB and rhtC, make the expression of these three genes by cuminic acid induction regulating controlling.
Have the following advantages compared with the expression system that the present invention adopts cuminic acid (cuminic acid) abduction delivering element and IPTG to induce: do not need to carry out specific genetic modification to Host Strains, the low control effects of background expression level is more rigorous, the quantitative adjustment of gene expression dose can be carried out, toxicity is not had to intestinal bacteria, and inductor is cheap, be applicable to suitability for industrialized production.
Beneficial effect of the present invention is, to knocking out metA gene, ilvA gene, lysA gene, tdh gene, repressor protein gene Expression element is imported in the bacterium that sets out of tdcC gene and sstT gene, Threonine arranges gene expression element and MUTATION Threonine operon Expression element outward, the recombinant bacterium obtained, cuminic acid induction can be carried out to Threonine production in fermentation logarithmic phase or stationary phase, improve the output of Threonine, the fermenting process that inductor controls the genetic expression of Threonine efflux protein is added by cuminic acid abduction delivering element, the production intensity of Threonine can be made to improve 10%-100%, glucose acid invert ratio improves 5%-50%, output increased 10%-150%, production amount of threonine can be increased to 70g/L-150g/L.Therefore in practice, can be used for fermentation using bacteria produce Threonine, easy to utilize, possess important industrial application value.
For the ease of understanding, below will be described in detail the present invention by specific embodiment.It is important to note that these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many changes of the present invention, to change concerning one of ordinary skill in the art be all apparent.
Accompanying drawing explanation
Fig. 1 is the structure of Threonine efflux protein gene Expression carrier pWY2167, pWY2168 and pWY2169
Fig. 2 is the double digestion checking of recombinant plasmid
Fig. 3 is the Threonine Fermentation conditional curve of the engineering bacteria that Dynamic controlling rhtC, rhtCA and rhtCAB express
Fig. 4 is chromosome mutation thrA
c1034Tthe cuminic acid induction type engineering bacteria of BC generates Threonine
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
As do not specialized in following embodiment, the conventional means that technique means used in embodiment is well known to those skilled in the art and commercially available common instrument, reagent, can see references such as the manufacturers instructions of " Molecular Cloning: A Laboratory guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent.
The following examples elaborate to the present invention, but do not limit the present invention.
The acquisition of embodiment 1, related strain and related plasmids
1, the structure of chassis engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT
On the basis of E.coli K12W3110, accumulation knocks out metA, ilvA, lysA, tdh, tdcC, sstT gene successively, primer sequence for gene knockout is as shown in table 1, final acquisition chassis engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT.
1) metA gene knockout
(can purchased from Japanese technological assessment institute Biological Resource Center (NITE BiologicalResource Center with E.coli K12W3110 bacterial strain, NBRC) genomic dna) is template, respectively with WY569 and WY570, WY571 and WY572 for primer carries out pcr amplification, obtain 503bpmetA upstream region and 473bp metA downstream area two DNA fragmentations, template is mixed into again with these two DNA fragmentations, with WY569 and WY572 for primer, by Overlap pcr amplification, obtain 976bpOverlap PCR junction fragment; Again Overlap PCR junction fragment and pKOV plasmid (can purchased from Addgene company) be connected, obtain recombinant plasmid, proceed to and treat in knock-out bacterial strain E.coli K12W3110, obtain the bacterial strain E.coliK12W3110 △ metA of metA gene knockout;
Use aligning primer WY583 and WY584 bacterium colony PCR outside metA gene to verify gene knockout, 1375bp is positive.
Concrete grammar is as follows: with the wild-type e. coli E.coli K12W3110 strain gene group DNA of extracting for template, respectively with WY569 and WY570, WY571 and WY572 for primer, EX Taq polysaccharase is used to carry out pcr amplification, PCR carries out as follows: 94 DEG C of sex change 30s(seconds), 54 DEG C of annealing 30s(seconds), and 72 DEG C extend 30s(second) (30 circulations).Two DNA fragmentations obtained by PCR after agarose gel electrophoresis separation and purification, then are mixed into template with these two DNA fragmentations, with WY569 and WY572 for primer, connect this two bar segment by the method for Overlap pcr amplification.Wherein, PCR carries out as follows: 94 DEG C of sex change 30s(seconds), 54 DEG C of annealing 30s(seconds), and 72 DEG C extend 60s(second) (30 circulations).Overlap PCR junction fragment after agarose gel electrophoresis separation and purification and pKOV plasmid are carried out double digestion with Bam HI/Not I respectively, connect after agarose gel electrophoresis separation and purification and use T4 ligase enzyme to connect, connect product conversion and turn competence to E.coli DH5 αization, select positive colony upgrading grain and sequence verification, the pKOV plasmid of checking exact connect ion Overlap PCR fragment is saved backup.By the above-mentioned carrier built respectively electricity be transformed in low yield E.coliK12W3110 competence, in 30 DEG C, 100rpm, in LB substratum after recovery 2h, according to the commodity guide of the pKOV plasmid of Addgene company, pick out the mono-clonal of the homologous recombination positive, use aligning primer WY583 and WY584 bacterium colony PCR outside metA gene to verify gene knockout, and confirm that the metA gene order on its karyomit(e) knocks out further by order-checking.
2) ilvA gene knockout
With the genomic dna of E.coli K12W3110 bacterial strain for template, respectively with WY577 and WY578, WY579 and WY580 for primer carries out pcr amplification, obtain 498bp and 530bp two DNA fragmentations, template is mixed into again with these two DNA fragmentations, with WY577 and WY580 for primer, by Overlap pcr amplification, obtain 1028bp Overlap PCR junction fragment; Again Overlap PCR junction fragment is connected with pKOV plasmid, obtains recombinant plasmid, proceed to and treat in knock-out bacterial strain E.coliK12W3110 △ metA, obtain the bacterial strain E.coli K-12W3110 △ metA △ ilvA of ilvA gene knockout;
Use primer WY587 and WY588 bacterium colony PCR to verify gene knockout, 1344bp is positive.
3) lysA gene knockout
With the genomic dna of E.coli K12W3110 bacterial strain for template, respectively with WY573 and WY574, WY575 and WY576 for primer carries out pcr amplification, obtain 507bp and 544bp two DNA fragmentations, template is mixed into again with these two DNA fragmentations, with WY573 and WY576 for primer, by Overlap pcr amplification, obtain 1051bp Overlap PCR junction fragment; Again Overlap PCR junction fragment is connected with pKOV plasmid, obtains recombinant plasmid, proceed to and treat in knock-out bacterial strain E.coliK12W3110 △ metA △ ilvA, obtain the bacterial strain E.coli K-12W3110 △ metA △ ilvA △ lysA of lysA gene knockout;
Use primer WY585 and WY586 bacterium colony PCR to verify gene knockout, 1302bp is positive.
4) tdh gene knockout
With the genomic dna of E.coli K12W3110 bacterial strain for template, respectively with WY598 and WY599, WY600 and WY601 for primer carries out pcr amplification, obtain 526bp and 632bp two DNA fragmentations, template is mixed into again with these two DNA fragmentations, with WY598 and WY601 for primer, by Overlap pcr amplification, obtain 1158bp Overlap PCR junction fragment; Again Overlap PCR junction fragment is connected with pKOV plasmid, obtain recombinant plasmid, proceed to and treat in knock-out bacterial strain E.coliK12W3110 △ metA △ ilvA △ lysA, obtain the bacterial strain E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh of tdh gene knockout;
Use primer WY602 and WY603 bacterium colony PCR to verify gene knockout, 1796bp is positive.
5) tdcC gene knockout
With the genomic dna of E.coli K12W3110 bacterial strain for template, respectively with WY476 and WY477, WY478 and WY479 for primer carries out pcr amplification, obtain 525bp and 536bp two DNA fragmentations, template is mixed into again with these two DNA fragmentations, with WY476 and WY479 for primer, by Overlap pcr amplification, obtain 1061bp Overlap PCR junction fragment; Again Overlap PCR junction fragment is connected with pKOV plasmid, obtain recombinant plasmid, proceed to and treat in knock-out bacterial strain E.coliK12W3110 △ metA △ ilvA △ lysA △ tdh, obtain the bacterial strain E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC of tdcC gene knockout;
Use primer WY497 and WY498 bacterium colony PCR to verify gene knockout, 1801bp is positive.
6) sstT gene knockout
With the genomic dna of E.coli K12W3110 bacterial strain for template, respectively with WY947 and WY948, WY949 and WY950 for primer carries out pcr amplification, obtain 683bp and 481bp two DNA fragmentations, template is mixed into again with these two DNA fragmentations, with WY947 and WY950 for primer, by Overlap pcr amplification, obtain 1164bp Overlap PCR junction fragment; Again Overlap PCR junction fragment is connected with pKOV plasmid, obtain recombinant plasmid, proceed to and treat in knock-out bacterial strain E.coliK12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC, obtain the bacterial strain E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT of sstT gene knockout;
Use primer WY949 and WY950 bacterium colony PCR to verify gene knockout, 1569bp is positive.
The primer that table 1. the present embodiment is used
2, plasmid pACYC184-P
pL-thrA
c1034Tthe acquisition of BC
Plasmid pACYC184-P
pL-thrA
c1034TbC is by MUTATION Threonine operon Expression element (P
pL-thrA
c1034TbC, sequence 1 is from 5 ' end 520-5654 position Nucleotide) insert expression vector pACYC184(and be purchased from NEB company, catalog number E4152S) Hind III and EcoR V restriction enzyme site between the carrier that obtains.
MUTATION Threonine operon Expression element P
pL-thrA
c1034TbC comprises MUTATION Threonine operon thrA
c1034TbC and its promotor P expressed of driving
pL, wherein, MUTATION Threonine operon thrA
c1034TbC be in sequence table sequence 1 from 5 ' end 688-5654 position Nucleotide; Promotor P
pLfor sequence in sequence table 1 is from 5 ' end 520-687 position Nucleotide.
Specific as follows:
With promotor P
pL(in sequence table, sequence 1 is from 5 ' end 520-687 position Nucleotide) is template, with WY842 and WY843 in table 2 for primer, uses Primer Star high fidelity polymerase enzymatic amplification promotor, reclaims with gel the promotor that test kit reclaims purifying amplification, for subsequent use;
With E.coli W3110 genome for template, respectively with WY914 and WY212, WY590 and WY213 is that primer PCR amplification obtains thrABC upstream and downstream fragment, cut glue reclaim thrABC upstream and downstream fragment and simultaneously with this two bar segment for template, obtained the threonine operon thrA of sudden change by the method (primer is WY914 and WY590) of overlap PCR
c1034TbC;
With Hind III and EcoR V restriction enzyme double digestion pACYC184 plasmid and the thrA cutting glue recovery respectively
c1034TbC, reuses gel and reclaims test kit recovery endonuclease bamhi; PACYC184 plasmid and the thrA of above-mentioned double digestion is connected with T4 ligase enzyme
c1034TbC gene fragment, is converted into DH5 α competent cell, screens positive transformant after Secondary Culture, send order-checking after extracting the checking of plasmid double digestion; Use Xba I and Hind III restriction enzymes double zyme cutting pACYC184-thrA
c1034Tthe promoter fragment P of BC plasmid and amplification
pL, the gene fragment after cutting with gel recovery test kit recovery enzyme.With the pACYC184-thrA that T4 ligase enzyme ligase enzyme is cut
c1034TbC plasmid and promoter fragment P
pL, change and go to DH5 α competent cell, after Secondary Culture, screen positive transformant, after the checking of upgrading grain double digestion, send order-checking to obtain correct recombinant vectors pACYC184-P
pL-thrA
c1034TbC.
Table 2 recombinant vectors pACYC184-P
pL-thrA
c1034TbC builds primer
3, chromosome mutation thrA
c1034Tthe engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT::P that BC obtains
pL-thrA
c1034TbC
E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT engineering bacteria karyomit(e) increases and removes feedback inhibition point mutation and the P controlling expression by strong promoter
pL-thrA
c1034TbC, obtains P
pL-thrA
c1034TbC is incorporated into the recombinant bacterial strain in karyomit(e) lysA site.
Recombinant bacterial strain E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT::P
pL-thrA
c1034TbC, for will containing P
pL-thrA
c1034Tthe recombinant bacterium that fragment (sequence 1) homologous recombination of BC obtains in the engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT of chassis.
Containing P
pL-thrA
c1034Tthe fragment of BC comprises lysA upstream homology arm (sequence 1 is from 5 ' end 1-519 position Nucleotide), P
pL-thrA
c1034TbC(sequence 1 is from 5 ' end 520-5654 position Nucleotide) and lysA downstream homology arm (sequence 1 is from 5 ' end 5655-6210 position Nucleotide).
Concrete grammar is as follows:
With the wild-type e. coli E.coli K12W3110 strain gene group chromosome of extracting for template, with WY970 and WY971 for primer, use EX Taq polysaccharase pcr amplification lysA upstream homology arm, amplification condition is: 94 DEG C of sex change 30s(seconds), 54 DEG C of annealing 30s(seconds), and 72 DEG C extend 30s(second) (27 circulations).The size that the above-mentioned pcr amplification of agarose gel electrophoresis separation and purification obtains is 543bp band, upstream homology arm and the pKOV plasmid of double digestion 543bp is carried out with Pst I/Bgl II, connect after agarose gel electrophoresis separation and purification and use T4 ligase enzyme to connect, connect product conversion and turn competence to E.coliDH5 αization, select positive colony upgrading grain and sequence verification, will verify that exact connect ion pKOV-lysA (upstream) plasmid saves backup.
With the wild-type e. coli E.coli K12W3110 strain gene group chromosome of extracting for template, with WY974 and WY975 for primer, use EX Taq polysaccharase pcr amplification lysA downstream homology arm, amplification condition is: 94 DEG C of sex change 30s(seconds), 54 DEG C of annealing 30s(seconds), and 72 DEG C extend 30s(second) (27 circulations).Use lysA downstream homology arm and the pKOV-lysA(upstream of Bam HI/Xba I double digestion 562bp) recombinant plasmid, connect after agarose gel electrophoresis separation and purification and use T4 ligase enzyme to connect, connect product conversion and turn competence to E.coli DH5 αization, select positive colony upgrading grain and sequence verification, will verify that exact connect ion pKOV-lysA (upstream)-lysA (downstream) plasmid saves backup.
With recombinant vectors pACYC184-P
pL-thrA
c1034TbC is template, carries out pcr amplification with primer WY978 and WY979, obtains the P of 5135bp
pL-thrA
c1034TbC.
Use the P of Bgl II and Bam HI double digestion
pL-thrA
c1034TbC, the digestion products obtained connects with the carrier framework of recombinant vectors pKOV-lysA (upstream)-lysA (downstream) 8560bp cut through same enzyme, connect product conversion and turn competence to E.coli DH5 αization, select positive colony upgrading grain and sequence verification, by pKOV-lysA (the upstream)-P of checking exact connect ion
pL-thrA
c1034TbC-lysA (downstream) plasmid saves backup.
Table 3 karyomit(e) lysA integrations P
pL-thrA
c1034Tthe structure primer of BC
According to the commodity guide of the pKOV plasmid of Addgene company, by pKOV-lysA (the upstream)-P built
pL-thrA
c1034TbC-lysA (downstream) plasmid electricity is transformed in the above-mentioned 1 chassis engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT obtained, pick out the mono-clonal of the homologous recombination positive, P is inserted in the lysA site confirmed on its karyomit(e) through order-checking
pL-thrA
c1034TbC fragment, successfully obtains recombinant bacterial strain E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT::P
pL-thrA
c1034TbC.
Embodiment 2, with plasmid form increase thrA
c1034Tthe structure of the cuminic acid induction type engineering bacteria of BC copy number
One, cuminic acid abduction delivering Threonine arranges the structure of the recombinant vectors of gene outward
1, repressor protein gene Expression element P
kmthe acquisition of-cymR
With Pseudomonas putidas F1(purchased from DSMZ company) genomic dna (DSM6899) be template, with WY899 and WY902 for primer, with Pseudomonas putidas genome for template, use PrimeSTAR polysaccharase to carry out pcr amplification and obtain cymR gene fragment; Sepharose reclaims the cymR band that PCR size is 775bp, continues with this cymR gene fragment for template, with WY900 and WY902 for primer PCR increases, and introducing portion P
kmpromotor obtains P
km1-cymR gene fragment; Sepharose reclaims P
km1-cymR PCR band, continues with P
km1-cymR gene fragment is template, with WY901 and WY902 for primer, uses PrimeSTAR polysaccharase to carry out pcr amplification, introduces complete P
kmpromoter sequence obtains the repressor protein gene Expression element P of 1132bp
km-cymR, reclaims test kit with gel and reclaims purifying, for subsequent use.
Repressor protein gene Expression element P
km-cymR(sequence 2) comprise repressor protein gene cymR(sequence 2 from 5 ' end 145-917 position Nucleotide) and the promotor P that drives it to express
km(sequence 2 is from 5 ' end 1-144 position Nucleotide).
2, Threonine arranges the acquisition of gene expression element outward
Use colibacillary strong promoter PT5 to be connected with controlling element Ocmt, after controlling element, connect Threonine efflux protein gene rhtC further, rhtA and rhtB, obtain Threonine and arrange gene expression element P outward
t5-Ocmt-rhtC, P
t5-Ocmt-rhtC-rhtA, P
t5-Ocmt-rhtC-rhtA-rhtB, makes the expression of these three genes by cuminic acid induction regulating controlling.
Threonine arranges gene expression element P outward
t5-Ocmt-rhtC(sequence 3), comprise Threonine and arrange gene rhtC(sequence 3 outward from 5 ' end 92-835 position Nucleotide), drive the promotor P of this genetic expression
t5(sequence 3 is from 5 ' end 1-59 position Nucleotide) and the controlling element Ocmt(sequence 3 that is positioned at therebetween are from 5 ' end 60-91 position Nucleotide).
Threonine arranges gene expression element P outward
t5-Ocmt-rhtCA(sequence 4), comprise Threonine and arrange gene rhtC(sequence 4 outward from 5 ' end 92-733 position Nucleotide) and rhtA(sequence 4 from 5 ' end 734-1706 position Nucleotide), drive the promotor P of this genetic expression
t5(sequence 4 is from 5 ' end 1-59 position Nucleotide) and the controlling element Ocmt(sequence 4 that is positioned at therebetween are from 5 ' end 60-91 position Nucleotide).
Threonine arranges gene expression element P outward
t5-Ocmt-rhtCAB(sequence 5), comprise Threonine and arrange gene rhtC(sequence 5 outward from 5 ' end 92-733 position Nucleotide), rhtA(sequence 5 is from 5 ' end 734-1637 position Nucleotide) and rhtB(sequence 5 from 5 ' end 1638-2342 position Nucleotide), drive the promotor P of this genetic expression
t5(sequence 5 is from 5 ' end 1-59 position Nucleotide) and the controlling element Ocmt(sequence 5 that is positioned at therebetween are from 5 ' end 60-91 position Nucleotide).
Concrete grammar is as follows:
With the genomic dna of wild-type e. coli E.coli K-12W3110 bacterial strain for template, carry out pcr amplification with primer WY847 and the WY883 in table 4, obtain the P of 835bp
t5-Ocmt-rhtC;
With the genomic dna of wild-type e. coli E.coli K-12W3110 bacterial strain for template, carry out pcr amplification with primer WY848 and the WY883 in table 4 and obtain rhtC fragment, carry out pcr amplification with primer WY849 and WY850 and obtain rhtA fragment, then with rhtA fragment and rhtC fragment for template, carry out with primer WY883 and WY850 the P that pcr amplification obtains 1706bp
t5-Ocmt-rhtC-rhtA.
With the genomic dna of wild-type e. coli E.coli K-12W3110 bacterial strain for template, carry out pcr amplification with primer WY848 and the WY883 in table 4 and obtain rhtC fragment, carry out pcr amplification with primer WY872 and WY873 and obtain rhtB fragment, carry out pcr amplification with primer WY849 and WY871 and obtain rhtA fragment.With rhtA fragment and rhtC fragment for template, carry out pcr amplification with primer WY849 and WY873 and obtain rhtA-rhtB fragment.Finally with rhtA-rhtB fragment and rhtC fragment for template, carry out with primer WY883 and WY873 the P that pcr amplification obtains 2342bp
t5-Ocmt-rhtC-rhtA-rhtB.
Table 4, primer sequence used
3, cuminic acid abduction delivering Threonine arranges the acquisition of recombinant vectors pWY2167, pWY2168, pWY2169 of gene outward
1) acquisition that Threonine arranges the intermediate carrier of gene expression element is outward connected with
By the P that above-mentioned 2 obtain
t5-Ocmt-rhtC, P
t5-Ocmt-rhtC-rhtA and P
t5-Ocmt-rhtC-rhtA-rhtB uses Eco RV and Eag I double digestion respectively, obtains the P of 835bp
t5-Ocmt-rhtC digestion products, 1706bp P
t5the P of-Ocmt-rhtC-rhtA digestion products and 2342bp
t5-Ocmt-rhtC-rhtA-rhtB digestion products; By above-mentioned digestion products respectively with the plasmid pACYC184-P obtained by 2 of embodiment 1 cut through same enzyme
pL-thrA
c1034Tthe skeleton of BC connects, and obtains intermediate carrier pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC, intermediate carrier
paCYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC-rhtA and intermediate carrier pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC-rhtA-rhtB.
By above-mentioned intermediate carrier through Eco RV and Eag I digestion verification, result is as Fig. 2 A, M:DNA marker; Swimming lane 1,3,5,7,9,11: plasmid band (contrast); Swimming lane 2 and 8: two band produced by Eco RV and Eag I double digestion plasmid 1 and 7, its medium and small band and goal gene fragment P
t5-Ocmt-rhtCAB size is coincide (2342bp), and this gene fragment and plasmid pACYC184-P are described
pL-thrA
c1034TbC successful connection; Swimming lane 10: two band produced by EcoRV and EagI double digestion plasmid 9, its medium and small band and goal gene fragment P
t5-Ocmt-rhtCA size is coincide (1706bp), and this gene fragment and plasmid pACYC184-P are described
pL-thrA
c1034TbC successful connection; Swimming lane 12: two band produced by EcoRV and EagI double digestion plasmid 11, its medium and small band and goal gene fragment P
t5-Ocmt-rhtC size is coincide (835bp), and this gene fragment and plasmid pACYC184-P are described
pL-thrA
c1034TbC successful connection, obtains intermediate carrier pACYC184-P respectively
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC, intermediate carrier pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC-rhtA and intermediate carrier pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC-rhtA-rhtB.
2) cuminic acid abduction delivering Threonine arranges the acquisition of the recombinant vectors of gene outward
By recombinant vectors pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC, recombinant vectors pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC-rhtA and recombinant vectors pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmt-rhtC-rhtA-rhtB cuts with Nru I and Bsu36I enzyme respectively, obtains pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmtthe skeleton of-rhtC9205bp, pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmtthe skeleton of-rhtC-rhtA10076bp and pACYC184-P
pL-thrA
c1034TbC-P
t5-O
cmtthe skeleton of-rhtC-rhtA-rhtB10712bp; By the 917bp repressor protein gene Expression element P that above-mentioned 3 skeletons obtain with cut through same enzyme above-mentioned 1 respectively
kmthe digestion products of-cymR connects, and obtains recombinant vectors pWY2167:pACYC184-P
pL-thrA
c1034TbC-P
km-cymR-P
t5-O
cmt-rhtC, pWY2168:pACYC184-P
pL-thrA
c1034TbC-P
km-cymR-P
t5-O
cmt-rhtCA and pWY2169:pACYC184-P
pL-thrA
c1034TbC-P
km-cymR-P
t5-O
cmtthe result schematic diagram of-rhtCAB(recombinant vectors as shown in Figure 1).
By above-mentioned recombinant vectors process Nru I and Bsu36I digestion verification, result as shown in Figure 2 B; Swimming lane 13-15:NruI and Bsu36I double digestion recombinant plasmid pWY2167, pWY2168 and pWY2169, all can produce and P
kmthe band (1132bp) that-cymR size is coincide, illustrates construction of recombinant plasmid success.
Recombinant vectors pWY2167 is by MUTATION Threonine operon Expression element (P
pL-thrA
c1034TbC, sequence 1 is from 5 ' end 520-5654 position Nucleotide) insert expression vector
pthe Hind III of ACYC184 and EcoR V restriction enzyme site, and P
t5-O
cmt-rhtC(sequence 3) insert Eco RV and the Eag I of expression vector pACYC184, and by P
km-cymR(sequence 2) insert the carrier that the Nru I of expression vector pACYC184 and Bsu36I restriction enzyme site obtain;
Recombinant vectors pWY2168 is by MUTATION Threonine operon Expression element (P
pL-thrA
c1034TbC, sequence 1 is from 5 ' end 520-5654 position Nucleotide) insert Hind III and the EcoR V restriction enzyme site of expression vector pACYC184, and by P
t5-O
cmt-rhtCA(sequence 4) insert Eco RV and the Eag I of expression vector pACYC184, and by P
km-cymR(sequence 2) insert the carrier that the Nru I of expression vector pACYC184 and Bsu36I restriction enzyme site obtain;
Recombinant vectors pWY2169 is by MUTATION Threonine operon Expression element (P
pL-thrA
c1034TbC, sequence 1 is from 5 ' end 520-5654 position Nucleotide) insert Hind III and the EcoR V restriction enzyme site of expression vector pACYC184, and by P
t5-O
cmt-rhtCAB(sequence 5) insert Eco RV and the Eag I of expression vector pACYC184, and by P
km-cymR(sequence 2) insert the carrier that the Nru I of expression vector pACYC184 and Bsu36I restriction enzyme site obtain.
Two, thrA is increased
c1034Tthe structure of the cuminic acid induction type engineering bacteria of BC copy number
The recombinant vectors pWY2167 prepared above-mentioned one, recombinant vectors pWY2168, recombinant vectors pWY2169 respectively electricity are converted into by 1 of the embodiment 1 chassis engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT obtained, and obtain recombinant bacterium A, recombinant bacterium B, recombinant bacterium C respectively.
The plasmid extracting recombinant bacterium A sends to order-checking, and this plasmid is pWY2167, the recombinant bacterium A called after E.coli △/pWY2167 containing this plasmid; For increasing thrA
c1034Tthe cuminic acid induction type engineering bacteria of BC copy number;
The plasmid extracting recombinant bacterium B sends to order-checking, and this plasmid is pWY2168, the recombinant bacterium B called after E.coli △/pWY2168 containing this plasmid; For increasing thrA
c1034Tthe cuminic acid induction type engineering bacteria of BC copy number
The plasmid extracting recombinant bacterium C sends to order-checking, and this plasmid is pWY2169, the recombinant bacterium C called after E.coli △/pWY2169 containing this plasmid; For increasing thrA
c1034Tthe cuminic acid induction type engineering bacteria of BC copy number.
After the same method, by expression vector pACYC184-P
pL-thrA
c1034TbC proceeds in the engineering bacteria E.coli K-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT of chassis, obtains control strain.
Embodiment 3, chromosome mutation thrA
c1034Tthe structure of the cuminic acid induction type engineering bacteria of BC
By the P that 2 of embodiment 1 obtain
t5-Ocmt-rhtC uses Eco RV and Eag I double digestion respectively, is connected respectively by above-mentioned digestion products, obtain intermediate carrier pACYC184-P with the pACYC184 skeleton cut through same enzyme
t5-O
cmt-rhtC.
Recombinant vectors pACYC184-P is cut with Nru I and Bsu36I enzyme
t5-O
cmt-rhtC, obtains the pACYC184-P of 4326bp
t5-O
cmt-rhtC skeleton; By the 917bp repressor protein gene Expression element P that this skeleton obtains with cut through same enzyme above-mentioned 1 respectively
kmthe digestion products of-cymR connects, and obtains recombinant vectors pACYC184-P
km-cymR-P
t5-O
cmt-rhtC, through order-checking, this recombinant vectors is by P
t5-Ocmt-rhtC(sequence 3) insert Eco RV and the Eag I of expression vector pACYC184, and by P
km-cymR(sequence 2) insert the carrier that the Nru I of expression vector pACYC184 and Bsu36I restriction enzyme site obtain.
By pACYC184-P
km-cymR-P
t5-O
cmt-rhtC electricity is converted into the recombinant bacterial strain E.coliK-12W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT::P obtained by 3 of embodiment 1
pL-thrA
c1034Tin BC, obtain recombinant bacterium D.
The plasmid extracting recombinant bacterium D sends to order-checking, and this plasmid is pACYC184-P
km-cymR-P
t5-O
cmt-rhtC, the recombinant bacterium D called after E.coli △/pACYC184-P containing this plasmid
km-cymR-P
t5-O
cmt-rhtC(karyomit(e)); For karyomit(e) being integrated sudden change thrA
c1034Tthe cuminic acid induction type engineering bacteria of BC.
Embodiment 4, dynamic regulation cuminic acid induction type engineering bacteria produce L-threonine
One, thrA is increased
c1034Tthe production L-threonine of the cuminic acid induction type engineering bacteria of BC copy number
1, the preparation of seed liquor:
Bacterial classification E.coli △/pWY2167, the E.coli △/pWY2168, the E.coli △/pWY2169 that are kept at-80 DEG C of cryopreservation tubes are lined LB flat board respectively, be placed in 37 DEG C of incubators and cultivate 12h, picking colony is seeded to the test tube containing 3mL LB substratum, be placed in 37 DEG C of shaking tables, 180rpm cultivates 12h.Get nutrient solution and be seeded to 500mL shaking flask containing 30mL seed culture medium by 3% inoculum size, be placed in 37 DEG C of shaking tables, 220rpm cultivates 12h, obtains E.coli △/pWY2167 seed liquor, E.coli △/pWY2168 seed liquor, E.coli △/pWY2169 seed liquor.
Above-mentioned seed culture medium is made up of the material of following final concentration and water: glucose 40g/L, ammonium sulfate 15g/L, potassium primary phosphate 2g/L, magnesium sulfate 2g/L, yeast powder 2g/L, Isoleucine 0.2g/L, calcium carbonate 15g/L, micro-mixed solution 5mL/L, paraxin 34mg/L.
Trace element mixed solution is made up of the material of following final concentration and water: FeSO
4.7H
2o, 10g/L; CaCl
2, 1.35g/L; ZnSO
4.7H
2o, 2.25g/L; MnSO
4.4H
2o, 0.5g/L; CuSO
4.5H
2o, 1g/L; (NH
4)
6mo
7o
24.4H
2o, 0.106g/L; Na
2b
4o
7.10H
2o, 0.23g/L; CoCl
2.6H
2o, 0.48g/L; 35%HCl, 10mL/L.
2, ferment
By above-mentioned 1 preparation various seed liquor press 3%(volumn concentration respectively) inoculum size access contain 2L fermention medium 7.5L fermentor tank in, fermentation culture, when strain growth starts stationary phase, (the 16th hour of fermented incubation time) adds inductor cuminic acid, makes its final concentration in fermentation system be 100 μm of ol/L; Obtain E.coli △/pWY2167 tunning, E.coli △/pWY2168 tunning, E.coli △/pWY2169 tunning respectively.
37 DEG C are maintained by heating jacket and cooling water control broth temperature in fermentor tank; Pass into air and provide dissolved oxygen, lead to oxygen and air Mixture if desired in the ratio of 1:1, rotating speed and dissolved oxygen signal cascade control dissolved oxygen and maintain 50%; Add 25% ammoniacal liquor regulation and control pH and maintain about 6.8.
Fermention medium is made up of the material of following final concentration and water: glucose 10g/L, ammonium sulfate 10g/L, potassium primary phosphate 2g/L, magnesium sulfate 2g/L, yeast powder 2g/L, micro-mixed solution 5mL/L, paraxin 34mg/L.
In above-mentioned seed and fermention medium, Isoleucine and paraxin are made into the liquid storage of 10g/L and 34g/L respectively with deionized water and anhydrous alcohol solution, filtration sterilization; Glucose is made into the liquid storage of 700g/L in 115 DEG C of autoclaving 15min.Other medium component 121 DEG C of high pressure steam sterilizations, seed culture medium sterilizing 15min, fermentation tank culture medium sterilizing 20min, the wherein independent sterilizing of magnesium sulfate, avoids producing precipitation.
In fermentation culture, manually set 7.5L
the rotating speed of 115NBS fermentation system built-in constant speed programmable control pump, constant speed adds glucose, regulates the constant speed of glucose liquid storage to add speed, make the glucose concn in fermentation system maintain 10 ± 5g/L according to the remaining sugar concentration of fermenting process.According to following index feed supplement equation, utilize BioCommand PlusBioProcessing software control fermentor tank built-in constant speed programmable control pump, realize index feed supplement.The thalli growth phase (fermentation culture 0-15 hour) add methionine(Met) (the total additional amount in fermentation system is 1g) according to the mass ratio index of 0.6:1.0:1.3, (the total additional amount in fermentation system is 1.7g to Isoleucine, please provide) and Methionin (the total additional amount in fermentation system is 2.2g), make thalline specific growth rate maintain 0.12-0.2h
-1, maintain thalli growth.
(/h), V
0and X
0represent Preliminary fermentation liquid long-pending (L) and dry cell weight (g/L) respectively; S
ithe concentration (g/L) of potassium primary phosphate or Isoleucine in feed supplement liquid storage, t represents the feed supplement time, and this parameter is the unique variable (h) of index feed supplement equation; Y
x/Sthe substrate yield (g/g) of biomass.
At different fermentation times respectively by E.coli △/pWY2167 tunning, E.coli △/pWY2168 tunning, E.coli △/pWY2169 tunning centrifuging and taking supernatant liquor, high pressure lipuid chromatography (HPLC) (HPLC) is used to detect amino acid, concrete grammar reference (Liu SW et al.Applied Microbiology and Biotechnology.2013,97 (2): 573-583.).
The control strain that embodiment 2 obtains is according to above-mentioned same method process.
Fermentation 48h, production amount of threonine result as shown in Figure 3, is added inductor cuminic acid induction efflux protein at 16h and is expressed, O represents control strain, CAB represents engineering bacteria E.coli △/pWY2169, and CA represents engineering bacteria E.coli △/pWY2168, and C represents engineering bacteria E.coli △/pWY2167.
Fig. 3 A is thalli growth situation, and Fig. 3 B is production amount of threonine; Can find out from Fig. 3 A, under the same conditions, the growth velocity of four kinds of engineering bacterias is basically identical, illustrates that the micro-background of cuminic acid inducible expression expresses the Threonine efflux protein produced, does not suppress thalli growth.
As can be seen from Figure 3B, compared with control strain, also can improve rate of producing acid at the micro-leakage expression of thalli growth stage efflux protein, and two (rhtCA) and three (rhtCAB) efflux protein leakage expressions faster than the rate of producing acid of the engineering bacteria only having rhtC leakage expression.And during Threonine efflux protein overexpression, specific growth rate and the Threonine production intensity in thalli growth stage can be reduced.Cuminic acid is induced, only the Threonine synthesis rate of E.coli △/pWY2167 bacterial strain of process LAN rhtC improves rapidly, and exceedes the synthesis rate of the E.coli △/pWY2168 of process LAN rhtC and rhtA and the E.coli △/pWY2169 of process LAN rhtC, rhtA, rhtB.
Ferment 48 hours, E.coli △/the pWY216(C of process LAN rhtC), the E.coli △/pWY2168(CA of process LAN rhtC and rhtA) and the E.coli △/pWY2169(CAB of process LAN rhtC, rhtA, rhtB) production amount of threonine be respectively 74.4g/L, 66.0g/L and 58.3g/L, be 51.3g/L than control strain O(production amount of threonine respectively) improve 45.0%, 28.7% and 13.6%.
Two, chromosome mutation thrA
c1034Tthe cuminic acid induction type engineering bacteria of BC generates Threonine
Seed culture method, inoculum size and ferment tank condition are the same, and fermentation results as shown in Figure 4, ferments 48 hours, control strain E.coli △/pACYC184-P
km-cymR and engineering bacteria E.coli △/pACYC184-P
km-cymR-P
t5-O
cmt-rhtC accumulates 18.5g/L and 24.1g/L Threonine respectively, makes production amount of threonine improve 30.3% by inducing the genetic expression of Threonine efflux protein in stationary phase.
Claims (10)
1. building a method for recombinant bacterium, is following A or B:
Method shown in A comprises the steps: that repressor protein gene Expression element and Threonine being arranged gene expression element importing outward sets out in bacterium, obtains recombinant bacterium;
Method shown in B comprises the steps: repressor protein gene Expression element, Threonine to arrange gene expression element and MUTATION Threonine operon Expression element outward and imports and set out in bacterium, obtains recombinant bacterium;
Described Threonine is arranged gene expression element outward and is comprised the control region that Threonine arranges gene outward, drives the Threonine inducible promoter of arranging genetic expression outward and be positioned at therebetween;
The promotor that described repressor protein gene Expression element comprises repressor protein gene and drives repressor protein gene to express;
The promotor that described MUTATION Threonine operon Expression element comprises MUTATION Threonine operon and drives MUTATION Threonine operon to express.
2. method according to claim 1, is characterized in that:
In method shown in B, described lead-in mode is following 1) or 2):
1) described repressor protein gene Expression element, described Threonine are arranged gene expression element and described MUTATION Threonine operon Expression element outward to be imported by recombinant vectors A and set out in bacterium, obtain recombinant bacterium;
2) described repressor protein gene Expression element and described Threonine being arranged gene expression element outward sets out in bacterium by recombinant vectors B importing, and set out in bacterium described in described MUTATION Threonine operon Expression element is imported by homologous recombination, obtain recombinant bacterium.
3. method according to claim 1 and 2, is characterized in that:
1), in, described repressor protein gene Expression element, described Threonine are arranged the carrier obtained in gene expression element and described MUTATION Threonine operon Expression element insertion expression vector outward by described recombinant vectors A;
2), in, described repressor protein gene Expression element and described Threonine are arranged gene expression element outward to insert the carrier obtained in expression vector by described recombinant vectors B;
Described homologous recombination is set out in bacterium to described with the form homologous recombination of the fragment containing MUTATION Threonine operon Expression element by MUTATION Threonine operon Expression element.
4. method according to claim 3, is characterized in that:
It is rhtC or rhtC and rhtB or rhtC, rhtB and rhtA that described Threonine arranges gene outward;
The inducible promoter that described driving Threonine arranges genetic expression is outward P
t5;
Described control region is Ocmt;
The promotor that described driving repressor protein gene is expressed is P
km;
Described MUTATION Threonine operon is MUTATION Threonine operon thrA
c1034TbC;
The promotor that described driving MUTATION Threonine operon is expressed is P
pL.
5. method according to claim 4, is characterized in that:
Described promotor P
t5nucleotides sequence be classified as sequence 3 or 4 or 5 in sequence table from 5 ' end 1-59 position Nucleotide;
The nucleotides sequence that described Threonine arranges gene rhtC is outward classified as the sequence 5 of sequence 3 in the sequence table sequence 4 in 5 ' end 92-835 position Nucleotide or sequence table in 5 ' end 92-733 position Nucleotide or sequence table from 5 ' end 92-733 position Nucleotide;
The nucleotides sequence that described Threonine arranges gene rhtA is outward classified as sequence 4 in the sequence table sequence 5 in 5 ' end 734-1706 position Nucleotide or sequence table from 5 ' end 734-1637 position Nucleotide;
The nucleotides sequence that described Threonine arranges gene rhtB is outward classified as sequence 5 in sequence table from 5 ' end 1638-2342 position Nucleotide;
The nucleotides sequence of described Ocmt is classified as sequence 3 or 4 or 5 in sequence table from 5 ' end 60-91 position Nucleotide;
Described promotor P
kmnucleotides sequence be classified as sequence 2 in sequence table from 5 ' end 1-144 position Nucleotide;
The nucleotides sequence of described repressor protein gene is classified as sequence 2 in sequence table from 5 ' end 145-917 position Nucleotide;
Described MUTATION Threonine operon thrA
c1034Tthe nucleotides sequence of BC is classified as sequence 1 in sequence table from 5 ' end 688-5654 position Nucleotide;
Described promotor P
pLnucleotides sequence be classified as sequence 1 in sequence table from 5 ' end 520-687 position Nucleotide.
6., according to described method arbitrary in claim 1-5, it is characterized in that:
The nucleotides sequence of described repressor protein gene Expression element is classified as the sequence 2 in sequence table;
The nucleotides sequence that described Threonine arranges gene expression element is outward classified as sequence 3 in sequence table or sequence 4 or sequence 5;
The nucleotides sequence of described MUTATION Threonine operon Expression element is classified as sequence 1 in sequence table from 5 ' end 520-5654 position Nucleotide;
The nucleotides sequence of the described fragment containing MUTATION Threonine operon Expression element is classified as the sequence 1 in sequence table.
7., according to described method arbitrary in claim 1-6, it is characterized in that:
The described bacterium that sets out is the sstT gene bacterium that obtains of totally 6 kinds of genes knocking out the metA gene of encoded homoserine succinyltransferase in bacterium, the ilvA gene of encoding Thr desaminase, the lysA gene of encoding D AP decarboxylase, the tdh gene of encoding Thr dehydratase, the tdcC gene of encoding Thr absorption and transport albumen and encoding Thr absorption and transport albumen;
Described bacterium is specially Escherichia bacteria, is especially specially intestinal bacteria.
8. the recombinant bacterium prepared by the arbitrary described method of claim 1-7.
9. generate a method for Threonine, comprise the steps: fermentation culture recombinant bacterium according to claim 8, and carry out cuminic acid induction at the second growth phase of described recombinant bacterium, collect the supernatant liquor of tunning, obtain Threonine;
Described second growth phase is exponential growth later stage, stationary phase or decline phase;
Described cuminic acid induction is specially 5-1000 μm of ol/L cuminic acid for adding final concentration in fermentation system;
The induction of described cuminic acid is especially specially that in fermentation system, add final concentration be 5-500 μm of ol/L cuminic acid.
10. a recombinant vectors is the recombinant vectors in the arbitrary described method of claim 1-7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410050917.2A CN104845995B (en) | 2014-02-14 | 2014-02-14 | A kind of method that dynamic regulation threonine arranges transporter gene Expression product L-threonine outside |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410050917.2A CN104845995B (en) | 2014-02-14 | 2014-02-14 | A kind of method that dynamic regulation threonine arranges transporter gene Expression product L-threonine outside |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104845995A true CN104845995A (en) | 2015-08-19 |
| CN104845995B CN104845995B (en) | 2018-11-13 |
Family
ID=53845983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410050917.2A Active CN104845995B (en) | 2014-02-14 | 2014-02-14 | A kind of method that dynamic regulation threonine arranges transporter gene Expression product L-threonine outside |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104845995B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400801A (en) * | 2015-11-18 | 2016-03-16 | 中国科学院微生物研究所 | ThrA gene mutant for relieving feedback inhibition and application thereof |
| CN110592084A (en) * | 2019-08-28 | 2019-12-20 | 内蒙古伊品生物科技有限公司 | Recombinant strain modified by rhtA gene promoter and construction method and application thereof |
| KR102183209B1 (en) * | 2019-09-09 | 2020-11-26 | 씨제이제일제당 주식회사 | Variants of L-threonine efflux protein and methods for producing L-threonine using them |
| CN113728105A (en) * | 2018-12-26 | 2021-11-30 | 大象株式会社 | L-amino acid-producing Escherichia coli mutant strain or Corynebacterium glutamicum mutant strain, and method for producing L-amino acid using same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597974A (en) * | 2003-09-17 | 2005-03-23 | 广东肇庆星湖生物科技股份有限公司 | Method for producing L-threonine by Escherichia coli |
| CN1898392A (en) * | 2003-12-24 | 2007-01-17 | 德古萨股份公司 | Process for preparing l-amino acids using strains of the enterobacteriaceae family |
| CN103865869A (en) * | 2014-04-03 | 2014-06-18 | 天津科技大学 | Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium |
-
2014
- 2014-02-14 CN CN201410050917.2A patent/CN104845995B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597974A (en) * | 2003-09-17 | 2005-03-23 | 广东肇庆星湖生物科技股份有限公司 | Method for producing L-threonine by Escherichia coli |
| CN1898392A (en) * | 2003-12-24 | 2007-01-17 | 德古萨股份公司 | Process for preparing l-amino acids using strains of the enterobacteriaceae family |
| CN103865869A (en) * | 2014-04-03 | 2014-06-18 | 天津科技大学 | Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium |
Non-Patent Citations (1)
| Title |
|---|
| 张雪 等: "含苏氨酸操纵子重组质粒的构建及其对大肠杆菌L-苏氨酸积累的影响", 《微生物学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400801A (en) * | 2015-11-18 | 2016-03-16 | 中国科学院微生物研究所 | ThrA gene mutant for relieving feedback inhibition and application thereof |
| CN105400801B (en) * | 2015-11-18 | 2018-08-17 | 中国科学院微生物研究所 | Release thrA gene mutation bodies and its application of feedback inhibition |
| CN113728105A (en) * | 2018-12-26 | 2021-11-30 | 大象株式会社 | L-amino acid-producing Escherichia coli mutant strain or Corynebacterium glutamicum mutant strain, and method for producing L-amino acid using same |
| CN113728105B (en) * | 2018-12-26 | 2024-05-28 | 大象株式会社 | L-amino acid-producing E.coli mutant strain or C.glutamicum mutant strain and method for producing L-amino acid using the same |
| CN110592084A (en) * | 2019-08-28 | 2019-12-20 | 内蒙古伊品生物科技有限公司 | Recombinant strain modified by rhtA gene promoter and construction method and application thereof |
| KR102183209B1 (en) * | 2019-09-09 | 2020-11-26 | 씨제이제일제당 주식회사 | Variants of L-threonine efflux protein and methods for producing L-threonine using them |
| WO2021049866A1 (en) * | 2019-09-09 | 2021-03-18 | 씨제이제일제당 (주) | L-threonine export protein variant and method for production of l-threonine using same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104845995B (en) | 2018-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107881186B (en) | Construction method and application of metabolic engineering escherichia coli strain for producing hydroxypropionic acid by using acetic acid | |
| WO2008119009A2 (en) | Materials and methods for efficient alanine production | |
| CN106086102B (en) | Engineering bacterium for producing trans-4-hydroxy-L-proline and construction method and application thereof | |
| CN105441497B (en) | Method for producing cadaverine by coupling microbial fermentation and microbial conversion | |
| CN105899664A (en) | Recombinant microorganism for improved production of fine chemicals | |
| CN102102086A (en) | L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof | |
| CN105051181B (en) | The preparation method of the increased recombinant microorganism of the generative capacity of 2,3-butanediol and the 2,3-butanediol using it | |
| CN105543214B (en) | Utilize the metabolic engineering coli strain construction method of acetic acid production succinic acid and application | |
| CN112063572B (en) | Recombinant escherichia coli capable of highly producing O-acetyl-L-homoserine and application thereof | |
| CN111705030B (en) | Escherichia coli genetic engineering bacterium capable of producing L-homoserine with high yield, construction method and strain | |
| CN104845995A (en) | Method for dynamic regulation and control of threonine efflux transport protein gene expression for production of L-threonine | |
| CN109456987B (en) | High-yield L-leucine related gene and engineering bacterium construction method and application | |
| CN107287144B (en) | Metabolically-modified bacillus subtilis biotransformation cell and preparation method and application thereof | |
| CN104894043A (en) | Engineering bacteria for producing gamma-aminobutyric acid and construction method and application thereof | |
| CN112592875A (en) | Homoserine producing strain and construction method and application thereof | |
| CN114957413B (en) | E.coli global regulatory factor cyclic adenosine receptor protein mutant, genetically engineered bacterium and application | |
| CN116024150A (en) | Genetic engineering strain for producing acetoin and construction method and application thereof | |
| CN116064345A (en) | Non-antibiotic genetic engineering bacteria for efficiently producing fucosyllactose and application thereof | |
| CN111500479B (en) | Construction and application of non-methanol-induced dual-promoter pichia pastoris engineering bacteria | |
| CN113817659B (en) | Method for preparing beta-alanine by metabolic engineering escherichia coli fermentation | |
| CN117844728B (en) | L-valine production strain and construction method and application thereof | |
| CN117004547B (en) | Genetically engineered bacterium for synthesizing cis, cis-muconic acid from glucose as substrate and application thereof | |
| CN113249281B (en) | Recombinant bacterium for producing phloroglucinol by using ethanol and construction method and application thereof | |
| WO2023092633A1 (en) | Recombinant bacterium for producing shikimic acid, construction method therefor, and application thereof | |
| CN116103213A (en) | Method for producing fumaric acid by metabolic engineering of escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |